Description Mostofourmechanisticunderstandingofhowhumancancercellsmigrateandinvadehasbeenobtainedby observingcellbehaviorinanartificial2 Denvironment.Althoughprogresshasbeenmadeusingthis approach,importantnewevidenceindicatesthatcellmigrationin2 Dsystemsdoesnotcompletely recapitulateeventsassociatedwithlocomotioninamorephysiologicalenvironmentusingreconstituted3 D matricesandtissueexplants.Workbyothersandnovelevidenceprovidedinthisresearchproposal demonstrateinvasivecellscanutilizeeitheramesenchymal typeofcellinvasionthatinvolvesformationofan elongatedinvadapodiaandaspindle shapedmorphologyoraprimitiveamoeboidmovementthatinvolves membraneblebbingthoughsmallholesintheextracellularmatrix.Thesebreakthroughfindingsprompted thehypothesisthatcellsarearmedwithdifferentinvasiveprogramsthatallowthemtotraversecomplex tissuesandcolonizeforeignsitesinthebody.Mostimportantlythoughthesefindingsindicatethat therapeuticpreventionofthisprocessinpatientswillrequireamultifacetedapproachthattargetsbothmodes ofcellinvasion.Itiscrucialthenthatweidentifyinvasivemechanismsutilizedbydisseminatingtumorcells invivosothattheappropriatetherapeuticagent(s)canbedesignedtocompletelyeradicatethespreadof cancerinpatients.However,tumorcellinvasionisacomplexanddynamicprocessthatinvolvestheintricate interplaybetweenthetumorcellsandtheremodelingvasculatureandstroma.Understandingthisprocessin vivohasbeendifficultbecauseithasnotbeenpossibletovisualizethisprocessinhighresolutioninlive animals.Toaddressthisproblem,wehavedevelopedanovelmodelofcancerprogressionthatutilizes humancancercellsgrowinginopticalclearzebrafishgeneticallyengineeredtoexpressgreenfluorescent proteininallbloodvessels.Usingthismodelanddualcolorhighresolutionconfocalmicroscopy,we discoveredthatthemetastaticgeneRhoCinducesarapidcellinvasionprocessthatfacilitatescellintravasation throughvascularopeningsinducedbyVEGFsecretion.Incontrast,mesenchymalcellinvasioninvolves formationofelongatedinvadopodiaandmembraneintegrationintothevascularwall,butnotcell intravasation.Ourgoalintheproposedworkistounderstandthesignalingmechanismthatcontrolamoeboid andmesenchymalinvasionascellsintravasateandhowthevascularporesforminresponsetoVEGF secretion.Basedonourpreliminaryfindingsandtheworkofothers,wehypothesizethatthemetastaticgene RhoCmediatesamoeboidinvasionthroughRhokinaseactivity(ROCK)andmyosinII mediatedcontractility. WealsohypothesizethatPI3Kharboringactivatingmutationsfoundinhumancancersinducesmesenchymal cellinvasionthroughactivationoftheFAK Src CAS Crk Racsignalingmodule,whichfacilitatesactin mediatedinvadopodialprotrusion.Wehypothesizethatthevascularporesformthroughdisruptionofcell celljunctions,whichisregulatedbysrcphosphorylationofVE cadherin.Therefore,ouroverallgoalisto examineindetailhowRhoCandmutatedPI3Ksignalingpathwaysregulatecancercellinvasionand intravasationandthemolecularsignalingmechanismsthatcontrolvascularporeformation. Cancercellsspreadthroughoutthebodybyinvadingintobloodvesselswheretheyarecarriedto distantorgansandformsecondarytumors.Workinthisproposalwilldeterminethemechanismof how cancer cells invade through the vessel wall utilizing high resolution confocal imaging of opticallytransparentzebrafishharboringmetastatichumancancercells.